American Society for Microbiology Intermountain Branch Annual Meeting April 10, 2010 8:00 AM --- Registration/Check-in, Poster set-up 9:15 AM --- Plenary

Session - ASMBL Lecture – Stanley Maloy, San Diego State University “From Cowpox to Mini-cells: New Approaches for the Delivery of Safe, Effective Vaccines” 10:30-10:45 --- Break 10:45-12:15 --- Concurrent sessions Session A: Virology, Dale Barnard, convener 10:45-11:15 Dale Barnard, Utah State “SARS-CoV Re-visited: Why Re-visit and What Progress Has Been Made?” 11:15-11:45 Michael Kay, University of Utah “D-peptide Inhibitors of HIV Entry” 11:45-12:00 Peter Shen, BYU “The structure of avian polyomavirus reveals pleiomorphic capsids and a putative location of the minor capsid protein VP4” 12:00-12:15 Daniel Hammond, BYU “Characterization of Herpes Simplex Virus Clinical Isolate Y3369” Session B: Gene expression/Physiology, Bill McCleary, convener 10:45-11:15 Joel Griffitts BYU “Two-component signaling in symbiosis” 11:15-11:45 John S. Parkinson, University of Utah “How a Bacterial Chemoreceptor Works” 11:45-12:00 Taylor Oberg, Utah State University “Physiological Stress Responses to Hydrogen Peroxide in Bifidobacterium animalis ssp. lactis” 12:00-12:15 Natalie Weber, Weber State University “Optimization of Medium M9 for Escherichia coli” 12:15-1:30 ---- Lunch and poster presentations 1:30-3:00 ---- Concurrent sessions Session C: Infection and Immunity, David Erickson, convener 1:30-2:00 Matthew Mulvey, University of Utah “Uropathogenic Escherichia coli: Dealing and Reeling with Nitrosative Stress” 2:00-2:30 Yongsheng Ma, Idaho State University “Virulence Factors and Their Regulation in Streptococcus pyogenes” 2:30-2:45 Britni Arlian, College of Idaho “Construction and characterization of an IsdA-cholera toxin A2/B chimera for use as a potential intranasal Staphylococcus aureus vaccine” 2:45-3:00 Lauren Zagieboylo, Brigham Young University “IgA ASC Accumulation to the Lactating Mammary Gland is Dependent on VCAM-1 and a4 Integrins” Session D: Applied and Environmental Microbiology (Don Breakwell, convener) 1:30-2:00 Frank Roberto, Idaho National Laboratories “Host and viral population dynamics and diversity within acidic Yellowstone hot spring environments” 2:00-2:15 Danielle Criddle, Utah State University “The Methylation of Mercury by Sulfate-Reducing Bacteria in the Great Salt Lake” 2:15-2:30 Blake Dahl, Weber State University “Molecular Characterization of Halophilic Bacteriophages Isolated from the Great Salt Lake” 2:30-2:45 Matthew Crook, BYU “Identification of a Phage Receptor in the Soil Bacterium Sinorhizobium meliloti” 2:45-3:00 Derek Bunch, Weber State “Effects of Salt Concentration on the Growth of Bacteriophage isolated from the Great Salt Lake, UT” 3:00-3:15 ---- Break 3:15-3:45 --- Student awards, Business meeting

Presentation Abstracts Oral Presentations 1. SARS-CoV Re-visited: Why Re-visit and What Progress Has Been Made? Dale Barnard Utah State University In 2003, a coronavirus (SARS-CoV) was identified as the causative agent of an emerging human infectious disease: severe acute respiratory syndrome (SARS). The outbreak of the SARS epidemic infected over 8000 people world wide, was reported in 30 countries, and led to the death of over 900 people with a fatality rate of at least 9.6%. Thus, in the early part of the last decade SARS posed a new threat for respiratory medicine and represented a challenge for antiviral drug development and administration. Yet the threat of coronaviruses related to SARS-CoV emerging to potentially cause severe disease in humans CoV ominously remains. This is due to the plasticity of the coronavirus genome (i.e., high frequency of homologous RNA recombination in a large genome) and the subsequent discovery of newly evolving animal coronaviruses that are closely related to the human SARS-CoV. In response to such a threat, there have been significant global efforts to develop vaccines and antiviral agents to prevent and/or to treat SARS-CoV infections. A number of animal models, primarily murine, have been developed for evaluating active in vitro anti-SARS-CoV agents as well as immune modulators. From studies done in lung virus replication murine and hamster models and in lethal mouse models, it has been found that interferon inducers, selected interferons, and a lectin, Urtica Dioica, are the most effective compounds for protecting mice from deleterious inflammatory responses, from death due to infection, and/or from virus replication in the lungs. Interestingly, IMP dehydrogenase inhibitors such as ribavirin either induced more lung virus replication or enhanced death. 2. D-peptide inhibitors of HIV Entry Michael Kay University of Utah During HIV-1 entry, the highly conserved gp41 N-trimer “pocket” region is transiently exposed and vulnerable to inhibition. Using mirror-image phage display and structure-assisted design, we have discovered protease-resistant D-amino acid peptides (Dpeptides) that bind the pocket with high affinity and potently inhibit viral entry. A trimeric version of one of these peptides (PIE12trimer) shows excellent potency and breadth against all major HIV subtypes, including Fuzeon-resistant strains. It also performs well in microbicide-specific antiviral assays and has a dramatically improved resistance profile compared to earlier generation Dpeptides and existing entry inhibitors. These results establish PIE12-trimer as a strong microbicide and therapeutic candidate. 3. The structure of avian polyomavirus reveals pleiomorphic capsids and a putative location of the minor capsid protein VP4 1,* 2 3 1 4 4 1 Peter S. Shen , Dirk Enderlein , Christian Nelson , Weston S. Carter , Masaaki Kawano , Li Xing , Robert D. Swenson , 5 5 4 3 2,6 1 Norman H. Olson , Timothy S. Baker , R. Holland Cheng , Walter Atwood , Reimar Johne , and David M. Belnap 1 Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA 2 Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, D-04103 Leipzig, Germany 3 Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, Providence, Rhode Island 02903, USA 4 Department of Molecular and Cellular Biology, University of California, Davis, California 95616, USA 5 Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, California 92093, USA 6 Current address: Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277 Berlin, Germany Avian polyomavirus (APV) is the causative agent of a fatal multi-systemic disease among various bird species. We used cryogenic electronic microscopy (cryo-EM) to solve the structure of APV at approximately 1-nm resolution and used sequence analysis and homology modeling to build a pseudo-atomic model of the major capsid protein (VP1). APV has a similar structure to mammalian polyomaviruses. However, APV VP1 has shorter C-terminal arms than all other known polyomaviruses, which we propose causes extensive size pleiomorphism in the T = 7d capsids. Treating APV with L-arginine was necessary to minimize particle aggregation and reduce size variation. Our cryo-EM based reconstructions also revealed electron densities plugged against the axial cavities of the VP1 pentamers, which is consistent with the location of minor capsid proteins VP2 and VP3; however, the densities are larger in APV capsids and may also represent densities of an additional minor capsid protein VP4, a protein only present in bird polyomaviruses. Treating JC polyomavirus with L-arginine produced reconstructions with empty axial cavities. Therefore, VP4 likely affects VP2 and VP3 binding to VP1 in APV. Our results support the model that VP4 is incorporated into the assembly unit of APV and facilitates particle assembly and genome packaging. 4. Characterization of Herpes Simplex Virus Clinical Isolate Y3369 Daniel Hammond, Daniel Clark, Brian Poole Brigham Young University Identification of Herpes simplex virus (HSV) isolates is achieved clinically through antigen detection by immunofluorescence (IF) assays. IF tests are both monoclonal antibody based (MAb), and polyclonal antibody based (PAb). While PAb can identify an unknown virus as HSV, only MAb can identify virus as HSV-1 or HSV-2. Isolate Y3369 tested positively with PAb, but was untypable using MAb assays. The purpose of this study was to identify and characterize the clinical isolate. To identify and

characterize the virus, DNA was extracted from purified virus and tested using PCR. PCR products were then sequenced and compared to HSV genomes acquired from GenBank. Virus was identified using a PCR test that detects the polymerase (pol) gene of HSV. Envelope proteins UL1, UL10, and UL22 were targeted as possible altered proteins, which caused the isolate to test negative for both HSV-1 and HSV-2. PCR testing showed that the isolate carried the HSV-1 pol gene. Sequencing showed a 97% agreement of PCR products for a common HSV-1 strain and the clinical isolate with published HSV pol genes. PCR testing for envelope proteins has identified equal DNA fragments from coding regions of envelope proteins UL1, UL10, and UL22. PCR has shown to be an accurate method for identification of HSV isolates. PCR tests for envelope proteins have shown similarities with common strains of HSV. Further testing of DNA is required to show how these strains differ. In addition infection assays will show if mutations in DNA have affected viral infectiousness or created resistance to anti-viral treatments. 5. Two-component signaling in symbiosis Joel S. Griffitts*, Rebecca E. Carlyon, Joanna L. Ryther, and Ryan D. VanYperen Brigham Young University, Department of Microbiology and Molecular Biology, Provo, UT Two-component signaling (TCS) is used by most bacteria to sense and respond to external stimuli. I will review the roles of TCS pathways in various host-microbe mutualisms, with an emphasis on the legume-rhizobium symbiosis. We have discovered a TCS pathway in Sinorhizobium meliloti that is required for symbiotic infection of its host plant alfalfa. The first component, FeuQ, encodes a sensor histidine kinase that is most likely integrated into the cytoplasmic membrane; the second component, FeuP, is a cytoplasmic transcriptional regulator that is controlled at the level of phosphorylation by FeuQ. The FeuQP pathway controls the secretion of a critical symbiotic oligosaccharide. We have more recently characterized a third protein, FeuN, which negatively regulates FeuQP-dependent gene expression. Our data suggest that FeuN may do this by switching FeuQ from a phosphodonor state to a phosphatase state. Interestingly, loss of FeuN function is lethal to S. meliloti, and explanations for this phenomenon will be discussed. While more remains to be learned about FeuN function, it contributes to an emerging theme of “third wheel” proteins that are being found to modulate TCS in diverse bacteria. 6. How a Bacterial Chemoreceptor Works John S. Parkinson Biology Department, University of Utah Motile bacteria seek optimal living habitats by following gradients of attractant and repellent chemicals in their environment. The signaling machinery for these chemotactic behaviors, although assembled from just a few protein components, has extraordinary information-processing capabilities. Escherichia coli, the best-studied model, employs a networked cluster of transmembrane receptors known as methyl-accepting chemotaxis proteins (MCPs) to detect minute chemical stimuli, to integrate multiple and conflicting inputs, and to generate an amplified output signal that controls the cell’s flagellar motors. Signal gain arises through cooperative action of chemoreceptors of different types. Studies of mutant chemoreceptors and their in vivo crosslinking patterns show that the receptor cluster is comprised of receptor signaling teams based on a trimer-of-dimers architecture. Signaling teams communicate through shared connections to cytoplasmic partner proteins (CheA, CheW). The structure of receptor clusters and how signaling teams communicate within the receptor array remain mysterious. The key to understanding transmembrane signaling by MCP molecules is the HAMP domain, a 50-residue motif that consists of two amphipathic helices (AS1, AS2) joined by a non-helical connector segment. In the homodimeric serine chemoreceptor, Tsr, the HAMP domain relays conformational signals between the periplasmic input domain and the cytoplasmic output domain. Two alternative HAMP structures have been described: HAMP(A), a tightly-packed, parallel, four-helix bundle; and HAMP(B), a more loosely-packed bundle, with an altered AS2/AS2' packing arrangement. Chemical stimuli probably modulate HAMP signaling by shifting the relative stabilities of these opposing structural states. Changes in AS2/AS2' packing, in turn, modulate output signals by altering structural interactions between output helices through heptad repeat stutters that produce packing phase clashes. A three-state, dynamic-bundle signaling model best accounts for the signaling properties of Tsr HAMP mutants. 7. Physiological Stress Responses to Hydrogen Peroxide in Bifidobacterium animalis ssp. lactis 1 1 2 2 1 T. S. Oberg , R. E. Ward , J. L. Steele , S. Ingham , J. R. Broadbent 1 Department of Nutrition and Food Sciences, Utah State University, Logan UT, USA 2 Department of Food Science, University of Wisconsin, Madison, WI, USA Recently there has been an increase in consumer and commercial interest in bifidobacteria as a probiotic in foods. Due to the anaerobic nature of bifidobacteria, oxidative stress can diminish viability of bifidobacteria during functional food production and storage. In this project, we examined the stress responses of Bifidobacterium animalis ssp. lactis BL04 and Bifidobacterium animalis ssp. lactis DSM10140 to hydrogen peroxide (H2O2). To determine the transcriptional stress response, each strain was grown to late log phase under anaerobic conditions with pH control. Each strain was then treated to a sub-lethal H2O2 concentration and incubated for 5 and 60 minutes. The mRNA was then isolated and cDNA was synthesized, fragmented and labeled. The samples were hybridized to an Affymetrix array and the resulting data analyzed using the limma/eBayes method. The BL04 strain had 158 significantly differentially expressed genes (P value < 0.05) after 5-minutes and 30 significantly differentially expressed genes after 60-minutes. The DSM10140 strain showed no significant differentially expressed genes at either time. The complete genome sequence of each strain was compared, and showed an incredible similarity with only 47 SNPs and four distinct insertion/deletion sites. Despite nearly identical genomes, these two strains clearly show a distinct phenotypic difference in their response to stress. One possibility for this difference could be a 54-bp deletion in the long chain fatty acid-coA ligase gene in the DSM10140 strain. To test this possibility, strains were grown as previously described, and the membrane fatty acids were isolated using the MIDI protocol. The samples were analyzed using a GC-MS and the fatty acids

were calculated as a percent of total. The profiles of the two strains were significantly different with the DSM10140 strain having a lower percentage of C14:0 and C16:0, and a higher percentage of C16:1n7 and C18:1n9. These differences could affect membrane fluidity and, thus, signal transduction during stress. This shows that the increased sensitivity to H2O2 in the DSM10140 strain could be caused by modification in this gene. 8. Optimization of Medium M9 for Escherichia coli Natalie Weber and William Lorowitz Department of Microbiology, Weber State University, Ogden, UT M9, a defined medium for growth of Escherichia coli, was optimized using statistical design of experiments (DOE) with absorbance as response variable. Screening with a factorial design revealed that Na2HPO4, KH2PO4, and NH4Cl had the greatest effects on growth. Steepest descent analysis suggested optimum values for Na2HPO4, KH2PO4, and NH4Cl were within the ranges 2.2 to 3.0 g/L, 1.45 to 1.50 g/L, and 0.34 to 0.38 g/L, respectively. Optimum values for these components were determined using response surface methodology. They were 3.0 g/L for Na2HPO4, 1.49 g/L for KH2PO4, and 0.35 g/L for NH4Cl. The values for these components in M9 medium are 6.0 g/L, 3.0 g/L, and 1.0 g/L, respectively. Comparisons of the modified medium with M9 and verification of the model are underway. This experiment was an excellent application of DOE, combining an educational experience with enhancement of a common medium for growth of E. coli. 9. Uropathogenic Escherichia coli: Dealing and Reeling with Nitrosative Stress Matthew Mulvey University of Utah, Division of Cell Biology and Immunology, Pathology Dept. The vast majority of urinary tract infections (UTIs) are caused by strains of uropathogenic Escherichia coli (UPEC), which can elicit a number of host proinflammatory and antimicrobial responses, including the production of reactive nitrogen species (RNS). By reacting with and damaging membrane proteins and other components of the bacterial envelope, RNS may modulate stress response pathways that allow the pathogens to better adapt to hostile host environments. A key way in which UPEC and other Gram-negative microbes respond to envelope stress is via the Cpx two-component system. Activation of the Cpx pathway transcriptionally regulates approximately 100 regulon members, many of which are involved in the folding and degradation of periplasmic and membrane proteins. One of the most highly responsive and activated regulon members is CpxP, a periplasmic regulator of the Cpx pathway. This talk will address the response of UPEC isolates to nitrosative stress, emphasizing a newly identified mechanism by which RNS post-transcriptionally repress CpxP expression. 10. Virulence Factors and Their Regulation in Streptococcus pyogenes Yongsheng Ma Idaho State University Group A streptococcus (GAS) causes many human diseases ranging in severity from milder infections such as pharyngitis and cellulitis to life-threatening necrotizing fasciitis/myonecrosis and streptococcal toxic shock syndrome (StrepTSS). Multiple extracellular toxins, alone and in combination, are thought to mediate these infections, yet the hierarchy of toxin regulators remains to be established with certainty. In the present study, a single insertion of transposon Tn917LTV1 into the chromosome of a StrepTSS-associated M-type 3 strain of GAS altered the expression of many extracellular proteins. Expression of the bicistronic genes speB/prsA in the wild-type strain was not detectable during exponential growth by Northern analysis, but was dramatically activated in the transposon mutant, demonstrating that SpeB production in the transposon mutant was no longer growth-phase dependent. In contrast, expression of nga/slo as well as many other proteins (e.g., immunogenic secreted protein, streptodornase, Mac/IdeS) was abruptly curtailed in the mutant. Chromosomal localization studies demonstrated a single transposon insertion into an open reading frame of a putative gene, identical to SpyM3_0606. Complementation of the transposon mutant with a plasmid containing SpyM3_0606 restored the wild-type toxin profile. This locus is unique to Streptococcus pyogenes and is distinct from other known global toxin regulators such as CsrR/S and Mga. Due to its varied effects on GAS extracellular toxin regulation, this gene has been designated vfr for virulence factor related. An understanding of Vfr-mediated toxin regulation may resolve unanswered questions regarding the global toxin regulatory network in S. pyogenes. 11. Construction and characterization of an IsdA-cholera toxin A2/B chimera for use as a potential intranasal Staphylococcus aureus vaccine 1 2 Britni M. Arlian and Juliette K. Tinker 1Department of Biology, The College of Idaho, 2112 Cleveland Boulevard, Caldwell, ID 83605 2Department of Biology, Boise State University, 1910 University Drive, Boise, ID 83725 Staphylococcus aureus represents a leading cause of nosocomial and community-acquired infection worldwide. With its increase in resistance to broad-spectrum antibiotics, effective treatment options have become limited, and S. aureus has emerged as a major public health threat. The ability of this gram-positive bacterium to cause a wide range of infections is the result of the expression of many virulence factors including IsdA, a surface protein essential for colonization of S. aureus on human nasal epithelial cells. In the present study, we constructed and characterized a potential S. aureus subunit vaccine using a cholera toxin fusion to IsdA. A plasmid was constructed to express a non-toxic cholera toxin (CT) A2/B chimera, containing the antigen IsdA from S. aureus, in transformed E. coli. Proper folding and composition of the IsdA-CTA2/B chimera was verified by SDS-PAGE and Western blot. The B subunit of the chimera was confirmed by GM1 ELISA to retain its affinity to bind CT’s native receptor, GM1. In vitro trafficking studies by confocal microscopy reveal that IsdA-CTA2/B binds to Vero cells and is

endocytosed in a manner similar to native CT. Female BALB/c mice were immunized intranasally with IsdA-CTA2/B, followed by one booster immunization 10 days post-priming. ELISA analysis of IsdA-specific antibody responses of feces and sera, and lymphocyte proliferation assay of mouse splenocytes stimulated with IsdA indicate intranasal immunization with IsdA-CTA2/B induced humoral IgA, systemic IgG, and cellular immunity. IgG induction by the chimeric molecule was superior to induction by IsdA antigen alone. Our findings suggest that further validation of the immunogenic properties of the IsdA-CTA2/B chimera could facilitate its use as a vaccine candidate in humans for prevention of S. aureus systemic infection or nasal carriage. 12. IgA ASC Accumulation to the Lactating Mammary Gland is Dependent on VCAM-1 and a4 Integrins Lauren Zagieboylo Elizabeth N. Low, Benjamin Martino and Eric Wilson Brigham Young University The homing and migration of IgA antibody secreting cells (ASC) to the lactating mammary gland is essential to the passive transfer of immunity from mother to nursing neonate. Antibody secreting cells, located within the lactating mammary gland, produce high levels of antigen specific IgA antibodies. These antibodies, which are subsequently transferred to the nursing neonate via breast milk, provide passive immune protection against antigens previously encountered by the mother to the nursing infant. The efficient homing and accumulation of lymphocytes is highly dependent on cellular adhesion molecules expressed on the vascular endothelium and their integrin ligands. Vasculature within the lactating mammary gland is known to express several adhesion molecules, including VCAM-1 and MAdCAM-1. However, the role of these molecules in vivo has not been previously described. Here we show that a4 integrins and VCAM-1 play essential roles in mediating the accumulation of IgA ASCs to the lactating mammary gland. Conversely, neither MAdCAM-1 nor its major ligand a4b7 are required for efficient IgA ASC accumulation to this tissue. 13. Host and viral population dynamics and diversity within acidic Yellowstone hot spring environments F.F. Roberto, M.M. Bateson, A.C. Ortmann, and M.J. Young Idaho National Laboratories Our team has been studying viruses and their hosts in several hot, acidic springs in Yellowstone National Park for the past decade. Our early work sought to uncover novel archaeal viruses in addition to those that had previously been reported for Sulfolobus by Zillig and colleagues. With the advent of so-called “next-generation” (or “now-gen”) sequencing technologies, we have expanded our study to consider the broader microbial and viral communities present, beyond those that we can cultivate and propagate. The relatively low biological complexity of high temperature (>80°C) low pH (<4.0) environments in Yellowstone hot springs provides an ideal opportunity to examine total viral and host diversity using metagenomic approaches. Our efforts have focused on two geographically separated hot springs, CHANN041, in the Crater Hills area of the Hayden Valley, and NL10, a hot spring along the north shore of Nymph Lake northwest of the Norris Geyser Basin. Our results indicate that each spring is dominated by a few crenarchaea and their associated viruses. The extensive sequence reads and relatively unbiased coverage possible with new sequencing technologies should enable us to reconstruct near-full length genomes of hosts and viruses in these less complex, extreme communities, to better understand their interactions with each other, and with the environment. 14. The Methylation of Mercury by Sulfate-Reducing Bacteria in the Great Salt Lake Danielle M Criddle* (Utah State University Department of Plants, Soils and Climate, Logan, UT), Giovanni Rompato (Utah State University Center For Integrated Biosystems, Logan, UT), Paul R Grossl (Utah State University Department of Plants, Soils and Climate, Logan, UT), J Jacob Parnell (Utah State University Center for Integrated Biosystems, Logan, UT) Great Salt Lake (GSL) is polluted with high levels of methylmercury (CH3Hg), a powerful neurotoxin. Since the lake is terminal pollutants tend to accumulate. This explains where mercury comes from but does not explain how it is getting converted into CH3Hg. This research aims to determine if sulfate reducing bacteria (SRB) are the primary methylators in GSL. The lake is highly saline (up to 10 times more saline than the ocean in some areas) and this high level of salt makes all the processes within the lake work differently, including the microbial community. Through this research we hope to optimize methods for use in GSL and determine the salt concentration that is optimal for sulfate reduction and mercury methylation. 15. Molecular Characterization of Halophilic Bacteriophages Isolated from the Great Salt Lake Ryan Hoggan, Blake Dahl* and Matthew J. Domek Weber State Univ., Ogden, UT Salinivibrio costicola is a halophilic bacterium originally isolated from salterns in Spain with one known halophage. It was recently isolated from the Great Salt Lake (GSL) and associated with two GSL halophage isolates (NS01 and CW02). To determine if NSO1 and CW02 are two unique halophage or variants of the same halophage, a modified protocol for isolation of λ halophage DNA was used to isolate the genetic material from NS01 and CW02. After cell lysis halophage were pelleted in PEG 8000, re-suspended, and treated with Proteinase K. Genetic material was purified by solvent extraction, and precipitated with ethanol. NS01 and CW02 genetic material was tested for degradation by DNase and RNase. Each halophage genome was then digested with EcoR1 and Hind III and their band patterns visualized by electrophoresis. DNase and RNase treatments revealed both halophage to be DNA viruses. Restriction digests with EcoR1 or Hind III each showed unique fragment patterns for each halophage. This suggests that NS01 and CW02 are different halophage, not just minor variants of the same halophage. Further investigation is needed to determine the genetic relationship between these two halophage and with S. costicola halophage isolated from the Spanish salterns.

16. Identification of a Phage Receptor in the Soil Bacterium Sinorhizobium meliloti Matthew B. Crook* and Joel S. Griffitts Brigham Young University, Department of Microbiology and Molecular Biology, Provo, UT Bacteriophages are ubiquitous in nature and are often adapted to infect a narrow range of bacterial hosts. There are several host-specific phages that are used to study the symbiotic nitrogen-fixing bacterium, Sinorhizobium meliloti, including ΦN3 and ΦM12. We isolated mutants of S. meliloti strain Rm1021 that were resistant to ΦN3 alone, to ΦM12 alone, and to both phages simultaneously. We then mapped the locations of the mutations in order to identify the putative phage receptors for each. To our surprise, mutations that conferred resistance to ΦN3 alone, to ΦM12 alone, and to both phages simultaneously all occurred in the same open reading frame: SMc02396, a predicted porin gene in the S. meliloti genome which is conserved in α-proteobacteria. We present a characterization of this gene. 17. Effects of Salt Concentration on the Growth of Bacteriophage isolated from the Great Salt Lake, UT Derek L. Bunch, Michele D. Zwolinski , Craig J. Oberg, Matthew J. Domek Weber State University, Ogden, UT 84408 The Great Salt Lake (GSL) is known for its unique environment. Its hypersalinity, with salt concentrations varying from eight to twelve percent in the south arm, inhibits the growth of most bacteria. Halophilic bacteria are abundant in the GSL, but not much is known about their unique ecology. Previously we obtained water samples from the south arm of the GSL and isolated several bacteriophage of halophilic hosts using a soft agar overlay technique. Bacteriophage for two specific hosts, Salinivibrio 50 (DB50) and Salinivibrio 36 (DB01), were isolated and amplified in broth culture then filtered at 0.2µm for further testing. Our hypothesis was to determine the effect of NaCl and MgSO4 concentration on the bacteriophage infectivity. Control media contained a 10:1 percent ratio of NaCl to MgSO4. Concentrations of 0% NaCl/0% MgSO4, 4%NaCl/0.4%MgSO4, 8%NaCl/0.8%MgSO4, 12%NaCl/1.2%MgSO4, and 16%NaCl/1.6%MgSO4 were tested. The two halophilic hosts were grown at the selected NaCl/MgSO4 concentrations with robust growth observed at 4%, 8%, and 12% and 16% NaCl. No growth was -3 observed at 0% NaCl. Bacteriophage infectivity was tested using the soft agar overlay technique with phage dilutions from 10 -7 8 9 8 to 10 . Results for phage DB50 were 3.5 x 10 pfu/ml at 4% NaCl medium, 2.3 x 10 pfu/ml at 8% NaCl, 2.1 x 10 pfu/ml at 12% 8 8 9 NaCl, and 3.0 x 10 pfu/ml at 16% NaCl. Results for phage DB01 were 1.6 x 10 pfu/ml at 4% NaCl, 5.0 x 10 pfu/ml at 8% 9 9 NaCl, 8.0 x 10 pfu/ml at 12% NaCl, and were greater than 10 at 16% NaCl. A large increase in phage number was observed between 4% NaCl and 8% NaCl while host growth was robust in both media. There appears to be a correlation between increased salt concentration and phage infectivity levels. Bacteriophage are important to the ecology of the GSL by playing a role in nutrient cycling, controlling bacterial populations, and seasonal changes in the GSL salt concentration may play a role in bacteriophage infectivity rates. Poster Presentations 18. The Resistance Profile of Human Papillomavirus Type 16 to Chemical Disinfectants. Jordan M. Meyers1, Eric Ryndock2, MJ Conway2, Kim L. O’Neill1, Craig M. Meyers2, and Richard R. Robison1 1Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT; 2Department of Microbiology and Immunology, Penn State University, Hershey, PA Human papillomavirus (HPV) represents one of the most prevalent sexually transmitted infections in the United States. The susceptibility of HPV to disinfection has not been described due to a lack of a suitable infectivity assay as well as the inability to produce sufficient amounts of HPV particles. Using a qRT-PCR infectivity assay for high-risk HPV16, we present the first disinfectant susceptibility profile for HPV16, the most common high-risk HPV type. We tested particles from two different propagation methods: native organotypically-derived virions and synthetic 293TT-derived quasivirions. Common clinical disinfectants tested included: ethanol, isopropanol, hypochlorite, alkaline glutaraldehyde, ortho-phthalaldehyde, peroxyacetic acid (PAA- based disinfectant), and a triple phenolic. Of these disinfectants, only the hypochlorite and PAA-based disinfectants were able to produce greater than a three log reduction of infectious virus for both particle types. All disinfectants were tested at relevant concentrations and at a 45 minute contact time. In addition, longer exposure times (24 and 48 hours) were tested for both aldehyde disinfectants. These data suggest that HPV has a very unique susceptibility profile relative to other nonenveloped viruses such as poliovirus and adenoviruses. For two disinfectants, the quasivirions were more susceptible than the native virions. Both alcohols were able to reduce infectivity of quasivirions at a 70% concentration and surprisingly, were equal to or more effective at a concentration of 95%. Both alcohols were ineffective against native virions, regardless of concentration. The results of these studies show that some disinfectants routinely used on medical instruments and devices may not be effective at inactivating native HPV, leaving open the possibility of iatrogenic transmission of HPV in a clinical setting. In addition, these data suggest that quasivirions do not have the same disinfectant resistance as more naturally-derived virions, suggesting that their use as surrogates in these types of studies may be problematic. 19. Inhibition of Severe Acute Respiratory Syndrome Coronavirus Replication in a Lethal SARS-CoV BALB/c Mouse Model by Stinging Nettle Lectin, Urtica Dioica Agglutinin (UDA) Yohichi Kumaki*, Miles K. Wandersee, Kevin W. Bailey, Aaron J. Smith, Craig W. Day, Jason R. Madsen, Donald F. Smee, Dale L. Barnard Institute for Antiviral Research, Utah State University, 5600 Old Main Hill, Logan, Utah 84322-5600

Urtica dioica agglutinin (UDA) was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. UDA is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity and inhibits viruses from Nidovirales in vitro. In the current study, groups of BALB/c mice were infected with 2 LD50 of virus and treated intranasally with UDA at the doses of 20, 10, 5 and 0 mg/kg/day for 4 days beginning at 4 h post virus exposure. Treatment with UDA at 5 mg/kg significantly protected mice against a lethal infection with mouse-adapted SARS-CoV (p<0.001), but did not significantly reduce virus lung titers. All mice receiving UDA treatments were also significantly protected against weight loss due to the infection (p<0.001). UDA also effectively reduced lung pathology scores. All mice receiving Poly IC:LC, the positive control drug, survived the infection (p<0.001). At day 6 after virus exposure, all groups of mice receiving UDA or Poly IC:LC had much lower lung weights than did the placebo-treated mice. Our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. 20. OCTADECYLOXYPROPYL ESTERS OF 3-HYDROXY-2-(PHOSPHONOMETHOXY)PROPYL NUCLEOSIDES SHOW ANTIVIRAL POTENCY AGAINST ADENOVIRUS IN VITRO J.D.Woolcott1, J.P. Roth3, T.Z. McLean1, N. Valiaeva2, J.R. Beadle2, K.Y. Hostetler2, D.L. Barnard1. 1Institute for Antiviral Research, Utah State University, Logan, UT, United States 2Division of Infectious Disease, University of California, San Diego, La Jolla, CA, United States 3 USDA Viral Diseases Research Unit, University of Georgia, Athens, GA, United States The majority of human adenovirus serotypes cause respiratory infections while other serotypes cause gastroenteritis, conjunctivitis, rash illness, and cystitis. Most adenovirus infections are mild, but a re-emerged serotype, adenovirus 14 (Ad14), was reported to cause severe and fatal pneumonia in rare cases of people of all ages. No antiviral compounds have been approved for the treatment of adenovirus infections and vaccines have been developed for only two serotypes, 4 and 7, to prevent acute respiratory disease (ARD) in military personnel. In this study, four nucleoside analog compounds, 2’,3’dideoxycytidine, ODE-HPMPA, ODE-HPMPC, and ODE-HPMPG, were evaluated against several adenoviruses. Neutral red uptake assays were used to test the potency of each compound in vitro. For adenovirus 1 (Ad1), the 50% antiviral efficacy values (EC50) ranged from 5 to 29 nM for the ODE-HPMPA/C/G compounds and 7 uM for 2’,3’-dideoxycytidine. For adenovirus 5 (Ad5), the EC50 values ranged from 21 to 300 nM for the ODE-HPMPA/C/G compounds and 17 uM for 2’,3’-dideoxycytidine. For Ad14, the EC50 values ranged from 4 to 310 nM for the ODE-HPMPA/C/G compounds and 13 uM for 2’,3’-dideoxycytidine. Virus yield reduction assays were used to validate the results. For Ad1, the 90% antiviral efficacy values (EC90) ranged from 3.5 to 9.2 nM for the ODE-HPMPA/C/G compounds and 3.6 uM for 2’,3’-dideoxycytidine. For Ad5, the EC90 values ranged from 1.6 to 9.2 nM for the ODE-HPMPA/C/G compounds and 6.2 uM for 2’,3’-dideoxycytidine. For Ad14, the EC90 values ranged from 6.5 to 20 nM for the ODE-HPMPA/C/G compounds and 12 uM for 2’,3’-dideoxycytidine. The 50% cell inhibitory concentrations for each compound on A549 cells were >3900 uM for 2’,3’-dideoxycytidine, 420 nM for ODE-HPMPA, 1400 nM for ODEHPMPC, and >7800 nM for ODE-HPMPG. Time of addition studies revealed that the potency of each compound was constant for up to 8 hours following adenovirus attachment. The results of this study support the further testing of these compounds as candidates for the clinical treatment of adenoviruses.  21. Loss of regulation in the Epstein Barr virus gene LMP1 by an IRF5 allele associated with risk for lupus Daniel Clark*, Brandon Henrie, and Brian D. Poole Dept. of Microbiology and Molecular Biology, Brigham Young University Systemic lupus erythematosis (SLE) is an autoimmune disease in which the body’s immune system creates antibodies against nuclear components such as DNA and leads to organ damage. One important environmental trigger is Epstein Barr virus (EBV). Especially among lupus patients, antibodies to EBV are cross-reactive to autoantigens, possibly contributing to lupus etiology. Genetic risk factors have been identified in a small number of genes including interferon regulatory factor (IRF)-5. We are studying the interactions of EBV and IRF5. IRF5 is involved in control of immune function in the interferon pathway, which normally works to control viral infections. The risk to SLE within IRF5 is due partly to the single nucleotide polymorphism (SNP) rs2004640 with the T allele conferring risk. We studied how IRF5 functions differently in nine gender and ethnicity matched sets, comparing homozygous risk to homozygous protective. We have discovered that the presence of the risk allele causes IRF5 to be less functional. IRF5 and a similar molecule, IRF7, act as transcription factors. IRF5, when functional, blocks IRF7 from increasing transcription of its target genes. One target of IRF7 is the EBV gene LMP1, which causes B cells to survive and proliferate. LMP1 levels were inversely correlated with IRF5 levels in cells with the protective allele at rs2004640 but in cells with the risk allele there was a positive correlation (r=0.74, p=0.02). LMP1 and two other targets of IRF7 were evaluated for levels of gene expression. All three genes were observed at higher mRNA expression levels in risk cells when compared to protective cells (from 1.3 to 1.5 average fold increase, p<0.05). Thus, IRF5’s ability to decrease gene expression of LMP1 is functionally impaired in rs2004640 risk allele-containing cells. Other targets of IRF7 showed similar loss of regulation by IRF5, establishing an important functional role for this risk factor to SLE. 22. Finding PAS Kinase Substrates Steven Sowa, Katherine Harris, Julianne H. Grose Brigham Young University PAS (Per-Arnt-Sim) kinase is a highly-conserved serine threonine kinase that acts in an unknown signal transduction pathway regulating glucose metabolism. The loss of this kinase protects mice on a high fat diet from obesity, insulin resistance, and high liver triglyceride levels, symptoms which are typically associated with type II diabetes. These discoveries prompted pharmaceutical companies to create inhibitors of PAS kinase in spite of the fact that the upstream regulators and the

downstream targets of PAS kinase remain largely unknown. We are utilizing several methods in Saccharomyces cerevisiae to determine these unidentified substrates of PAS kinase. A yeast two hybrid screen has been started to identify PAS kinase binding partners using a yeast genomic library. One advantage of performing this substrate screen in yeast is that yeast PAS kinase has known activating conditions, namely growth on non-fermentable carbon sources such as galactose or raffinose. Therefore, we can perform the screen for substrates in both activating and non-activating (growth on glucose) conditions and compare results. One challenge with the yeast two hybrid screen is that the interactions of substrates with PAS kinase are expected to be transient, so they may be difficult to detect. In order to enrich our screen, we are working on a substrate-trapping system to increase PAS kinase’s affinity for its substrates. In an effort to augment our yeast two hybrid method for identifying substrates, we are also conducting in vivo tests to confirm a suspected substrate of PAS kinase, STM-1. In combination, these methods will broaden our understanding of PAS kinase’s targets and function. Since PAS kinase also has a human homolog, this research could greatly expand our knowledge of obesity and type II diabetes in humans. 23. The Role of PAS Kinase in Regulating Cell Wall Biosynthesis of Yeast Kevin Wiest and Julianne Grose Brigham Young University PAS kinase is a highly conserved member of the nutrient-sensing protein kinases that has been researched in correlation with its role in the development of type II diabetes and obesity. In studies with mice, PAS kinase-deficient mice were found to have an increased metabolic rate, were protected from insulin resistance and high liver triglyceride levels, and were resistant to highfat diet-induced obesity. Currently, its role in the signal transduction pathway regulating glucose metabolism is unknown. There are no known mammalian substrates of PAS kinase, but UDP-glucose pyrophosphorylase (Ugp1) has been identified as a substrate in yeast models. This enzyme produces UDP-glucose, the immediate glucose donor for both glycogen and cell wall glucan biosynthesis. It has been shown that PAS kinase phosphorylation of Ugp1 has no effect on the catalytic activity of Ugp1, but instead, it changes the destination of its product favoring cell wall biosynthesis at the expense of glycogen biosynthesis. My project explores the function of phosphorylated Ugp1 in cell wall biosynthesis. In previous studies, tagging Ugp1 caused Ugp1 to be unphosphorylatable, making it difficult to study phospho-Ugp1 function. Thus, we have designed a genetic screen to isolate a mutant from of Ugp1 that is trapped in a phosphorylated conformation. Through this screen, we have found three mutations that are plasmid-dependent and appear to trap Ugp1 in the phosphorylated conformation. Since Ugp1 changes its conformation when phosphorylated, this conformation can be detected by chromatography as well as trypsin digest. These mutations will also allow us to localize phospho-Ugp1 as well as to identify phosphorylated Ugp1 associated proteins, furthering our understanding of the function of PAS kinase in regulating the biosynthesis of the cell wall. By studying the different roles of PAS kinase in a yeast model, a greater understanding of its pathway in mammals will be achieved. 24. Functional Coupling as a Tool for Finding NAD(P)–related Genes Chris Johnson, Julianne H. Grose Brigham Young University Evidence is accumulating that most of the degenerative diseases that afflict humanity originate from deleterious free radical reactions. Cancer, diabetes, neurodegeneration, inflammatory syndromes, and infectious disease can all be caused by irreversible cellular damage due to reactive oxygen species, or free radicals. The concentrations of NAD/NADH and NADP/NADPH determine the cellular redox state, lending to the fact that altered regulation and degradation of these important molecules could impair the cellular redox state and thus contribute to the mechanisms of pathogenesis in the afore mentioned diseases. Currently not much is known about the synthesis and regulation of NAD/NADP, and since these are essential molecules, novel approaches must be taken in order to gain a better understanding of them. The idea of Functional Coupling entails genes with similar function, or genes that operate in the same pathway, being paired together on the bacterial chromosome. This idea has led to the notion of finding unknown genes that operate in the regulation and synthesis of NAD/NADP through bioinformatic studies with bacteria. The use of bioinformatics has resulted in identifying several wellconserved genes of unknown function that appear to be involved in NAD(P) metabolism and we are currently investing their function in the cell. 25. The effects of NADPH Biosynthesis on Cardiomyopathy Kent Jarvis, Jonathan Neubert, and Julinne Grose Brigham Young University Mutation in the human heat shock alpha B-crystallin protein CryAB (hR120GCryAB) causes a rare hereditary disease that results in cardiomyopathy. This mutation causes protein aggregates to form in the heart and ultimately leads to heart failure. Studies in mice show that the mutation causes cardiac myopathy by a “gain of function mechanism” (Rajasekaran et al., Cell 130: 427-439, 2007). In addition, development of cardiomyopathy is dependent on overexpression of glucose-6-phosphate dehydrogenase (G6PD). G6PD is the main enzyme that increases levels of NADPH and is the predominate source of reductive power in cell biosynthesis and repair reactions. Together, the coenzymes NAD(H) and NADP(H) participate in over 300 cellular redox reactions. These reactions are essential to cellular metabolism, including enzymatic activity in glycolysis, various reactions within the TCA cycle, biosynthesic reactions and especially the electron transport chain. Misregulation of NAD(P)(H) levels has been reported in a wide variety of diseases, including diabetes, cancer and neurodegenaration. In order to understand the mechanism of R120GCryAB pathogenesis, we are running a yeast two-hybrid to identify proteins that interact with both the wild type and the mutant CryAB. We are also screening other genes (such as G6PD) that are associated with

CryAB cardiomyopathy. Knowing which proteins interact with these genes will help us better understand how mutant CryAB and misregulation of NADPH levels leads to cellular disease. 26. Identification of Yersinia pseudotuberculosis genes that affect congo-red binding Ryan Stewart and David Erickson Department of Microbiology and Molecular Biology, Brigham Young University The plague bacterium Yersinia pestis recently evolved from the enteric pathogen Yersinia pseudotuberculosis, during which time it has adopted a flea-borne lifestyle. Y. pestis forms a biofilm in the flea digestive tract to enhance transmission, whereas Y. pseudotuberculosis does not. Biofilm requires the hms genes, which direct the production of an N-acetylglucosamine extracellular matrix (ECM) that absorbs Congo-red (CR) dye, and Y. pestis typically forms pigmented colonies on CR agar. The Hms proteins of Y. pestis and Y. pseudotuberculosis are identical, yet most Y. pseudotuberculosis isolates are non-pigmented on CR agar. Despite extremely high overall similarity, Y. pestis has lost the function of ~120 genes that appear intact in Y. pseudotuberculosis. Among these non-functional genes are putative transcriptional regulators, and other genes that could affect the production or stability of the Hms ECM. Inactivation of these genes in Y. pestis could have enhanced biofilm and flea-borne transmission. We inserted functional copies (from Y. pseudotuberculosis) of six predicted regulatory genes into Y. pestis on plasmids. We also replaced the Y. pseudotuberculosis functional copies of three genes with non-functional Y. pestis versions. The CR binding phenotypes were not changed by any of these changes. We also used random transposon mutagenesis to identify mutants of Y. pseudotuberculosis that bind CR dye. We found four separate mutants that stably express a CR-positive phenotype. We mapped the insertion sites for three of the mutants, which included a putative LuxR-type regulator, the stationary-phase response protein Dps, a SecY interacting protein, and a putative polysaccharide deacetylase gene. Three of these mutants also exhibited enhanced swimming and swarming motility in comparison to wild-type Y. pseudotuberculosis. The polysaccharide deacetylase of Y. pestis has a single amino-acid change in the conserved deacetylase domain compared to the Y. pseudotuberculosis version, which may affect its activity. Future work will focus on characterizing the function of these genes in both Y. pestis and Y. pseudotuberculosis biofilm and flea infection. 27. Detecting protein-protein interactions with a Tat-dependant beta-lactamase reporter Casey M. Crum*, Rachel Jolley, and James D. Palmer, Joel Griffitts Brigham Young University, Department of Microbiology and Molecular Biology, Provo, UT We are designing a system with which to detect protein-protein interactions using ampicillin resistance similar to the recent work by Waraho and DeLisa at Cornell. We have developed a novel scaffold protein (GB1, derived from the streptococcal protein G) that will allow us to randomize specific domains without compromising the structural integrity of the folded protein. Using this two-plasmid system, we can quickly screen through large libraries of randomized GB1 and find variants that exhibit high binding affinity to a protein of interest. Transcriptionally fused to GB1 is ssTorA, a signal sequence that directs folded proteins into the periplasm via the twin arginine translocation (Tat) pathway. The protein of interest has a transcriptional fusion to beta lactamase, which provides ampicillin resistance only when expressed in the periplasm. The functionality of the system can be tested by substituting Fos and Jun, two proteins that are know to dimerize well, in place of GB1 and the protein of interest. It is only when Fos and Jun dimerize that the four-protein complex is directed to the periplasm via ssTorA and confers antibiotic resistance to the cell due to the actions of beta lactamase . Once we have demonstrated the functionality of the system, it can be used to find GB1-based antibodies for any protein of interest. 28. A New Approach to Recombination-Based in vivo Expression Technology (RIVET) in Sinorhizobium Meliloti Clarice Harrison* and Joel Griffitts Brigham Young University, Department of Microbiology and Molecular Biology, Provo, UT We are currently developing a versatile version of recombination-based in vivo expression technology (RIVET). It is based on invivo expression technology (IVET), which is commonly used to trap promoters by placing a reporter gene near a promoter of interest. RIVET systems take this one step further by inserting a gene by a promoter that when expressed will cause sitespecific recombination in another cassette already in the genome. This irreversible recombination is useful in assaying when a promoter is induced. Our modified RIVET system features a promoter trap made of tetracycline resistance and cre genes bounded by transposon repeats. It also has an excision cassette that has a constitutive promoter reading into a gentamicin resistance gene flanked by loxP sites followed by a spectinomycin resistance gene, and gus both promoter-less. When cre lands behind a promoter, cre will also be expressed which will lead to excision of the gentamicin gene and the expression of spectinomycin resistance and gus. In this project we used this system to find promoters that are induced only by addition of certain chemicals to a bacteria’s medium. We also hope to use this system to delete selected genes from Sinorhizobium meliloti to better visualize the effects of loss in growing cells. 29. Two-component signaling in symbiosis Ryan D. VanYperen, Rebecca E. Carlyon, Joanna L. Ryther, Joel. S. Griffitts Brigham Young University, Department of Microbiology and Molecular Biology, Provo, UT Bacteria use two-component systems to sense the environment and respond appropriately to external conditions. We have discovered in Sinorhizobium meliloti a two-component system which is required for symbiotic infection with an alfalfa host. FeuQ encodes a trans-membrane sensor kinase which appears to activate in response to osmolaric conditions. FeuP encodes a transcriptional response regulator under the control of FeuQ. FeuP is activated by FeuQ by phosphorylation. A third

regulatory component, FeuN, is involved in the negative regulation of this two-component system. Our data shows that FeuN regulation occurs independently from environmental conditions. Our data suggests that FeuN localizes to the periplasm, and therefore likely interacts directly with FeuQ. This interaction may cause FeuQ to change to a phosphotase state instead of a phospho-donor for FeuP. We are currently using biochemical methods to characterize the interaction between FeuN and FeuQ. 30. Determination of Escherichia coli K12 Lifespan Hyrum Gillespie, Jacob Parnell, Giovanni Rompato Utah State University, Center for Integrated Biosystems Because bacteria reproduce by asexual binary fission, calculating the life span has proven elusive. Nevertheless, the bacterial life span and death rates are as important as growth rates. We hypothesize that by inhibiting cell division, we can determine the lifespan of bacteria. In order to test our hypothesis, we arrested cell division in E. coli K12 using nalidixic acid (NAL), a quinolone that specifically targets and blocks DNA replication. We optimized the concentration of NAL to inhibit cell division without causing cytotoxicity. This was confirmed by quantitative expression of cell division genes and stress response genes shown to respond to NAL. We hypothesize that temperature dependent death rates of E. coli with this concentration of NAL correlate with bacterial lifespan. 31. Time-course analysis of the Shewanella amazonensis SB2B proteome in response to osmotic shock 1 2 3 3 Ashley Williamson , Stephen Callister , Giovanni Rompato , Jacob Parnell 1 Logan High School, Logan, UT 84321 2 Biological Separations and Mass Spectrometry, Pacific Northwest National Laboratory, Richland, WA 99352 3 Center for Integrated BioSystems and Department of Biology, Utah State University, Logan, UT 84322 Organisms in the genus Shewanella have become models for response to environmental stress. One of the most important environmental stresses is change in osmolarity. In this study, we experimentally determine the response mechanisms of Shewanella amazonensis SB2B during osmotic stress. SB2B was exposed to 0.85M NaCl, corresponding to a 50% reduction in growth rate, and the time-course response was analyzed using advanced proteomic technology, gene expression methods, and phenotypic characterization. The immediate response to salt stress involves induction of proteins involved in sensing the environment and repression of replication and cell division. The intermediate response is induction of proteins involved in branched chain amino acid degradation, followed by re-induction of replication and cell division proteins. Earlier studies suggested that high salt represses motility as confirmed in our study. Salt has also been associated with a change in the membrane fatty acid composition (due to induction of branched chain amino acid degradation pathways); however we show this is not the case as proteins and genes involved with fatty acid degradation are not induced and no change in the fatty acid profile occurs in SB2B as a result of osmotic shock. 32. Construction of a West Nile Virus vaccine candidate by fusion to Vibrio cholerae cholera toxin. Jie Yan and Juliette Tinker Department of Biology, Boise State University, Boise, Idaho 83725, USA. West Nile Virus (WNV) is a mosquito-borne flavivirus that has spread widely throughout the world, causing frequent outbreaks of disease in Africa, Asia, Europe, the Middle East, and more recently in the United States. The current estimates are that more than 30,000 cases of West Nile fever and neuroinvasive disease have occurred in the U.S., with greater than 1000 deaths. While vaccines are currently available for horses, they are not suitable for human use. Several human vaccine candidates are in development; however they often rely on the injection of attenuated viral delivery systems and are not amenable to oral or mucosal delivery. In addition, these vaccines may be unsuitable for immunocompromised individuals who are the most likely to suffer severe WNV symptoms. The protein toxin produced by the bacterium Vibrio cholerae, cholera toxin (CT), is a powerful vaccine adjuvant that is able to induce immune responses to co-administered antigens from mucosal surfaces. We cloned the WNV envelope domain III (E-DIII) and pre-membrane (prM) proteins into vectors expressing the non-toxic CTA2/CTB domains of cholera toxin to produce chimeric vaccine candidates. These constructs were expressed in Escherichia coli and purified by Dgalactose affinity chromatography from the culture supernatant. SDS-PAGE analysis and western blot were performed to characterize the expression and composition of these fusion proteins. Future work will involve the pre-clinical characterization of these potential WNV vaccine candidates. 33. Gene expression analysis of Xenopsylla cheopis fleas reveals a role for reactive oxygen species in controlling Yersinia pestis infection Nick Whipple, Wei Zhou, Christopher Conley and David Erickson Department of Microbiology and Molecualr Biology, Brigham Young University Fleas are vectors for a number of pathogens, including the plague bacterium Yersinia pestis. The physiological and immunological factors that govern interactions between fleas and Y. pestis are not understood. There is also a paucity of genome, transcriptome, and proteome data available for molecular studies of flea transmission. We conducted a transcriptome analysis of Xenopsylla cheopis (the Oriental rat flea) using 454 pyrosequencing of cDNA fragments derived from RNA isolated from whole fleas. The individual reads were assembled into 75,684 contigs. BLAST analysis of the individual reads most often yielded matches closest to sequences from the flour beetle Tribolium castaneum. We also generated cDNA libraries through

suppression subtractive hybridization that represent X. cheopis genes that are upregulated in response to oral or hemocoel Y. pestis infection, 24 or 48 hours after infection. We identified many genes with predicted functions in pathogen recognition, response, and containment. In particular, we saw several genes that likely affect the production of reactive oxygen species (ROS). Consistent with this observation, measurements of peroxide levels in dissected midguts were higher from infected compared with uninfected fleas. We cloned and completely sequenced a dual oxidase (DuOx) cDNA that may contribute to the rise in ROS levels. DuOx mRNA was detected in midgut tissues, and transcript levels were 6-fold higher following infection with Y. pestis. We inhibited ROS production by treating fleas with the antioxidant N-acetylcysteine, and found that infection rates and bacterial loads were higher in the treated fleas compared with untreated controls. Taken together, our results suggest a role for ROS in limiting survival of Y. pestis in fleas, and that bacterial factors that enable ROS survival may be important transmission factors. 34. Investigating the Function of the Yersinia Murine Toxin in Flea Infections Bria Bird, Sarah Gehen, and David Erickson Brigham Young University Yersinia pestis, the causative agent of plague, is transmitted by fleas such as the Oriental rat flea Xenopsylla cheopis. Very little is known about the Y. pestis factors that are important in establishing a transmissible infection in fleas. The Yersinia Murine Toxin (Ymt) is an intracellular phospholipase enzyme that is required for Y. pestis to survive inside the midgut after fleas take an infectious blood meal. The basis for the Ymt requirement is unknown. Since ymt mutants of Y. pestis can survive in fleas that feed on washed red blood cells without serum, yet ymt mutants grow in serum in vitro, it is likely that a component of serum digestion kills Y. pestis. Serum contains thiocyanate (SCN-) ions and we have previously shown that flea midguts contain high levels of hydrogen peroxide (H2O2). SCN- and H2O2 can be converted by peroxidases into the potent antimicrobial hypothiocyanate (OSCN-). We tested the in vitro susceptibility of Y. pestis wild-type and ymt mutant strains to OSCN- and found that both were equally susceptible. We also found that adding SCN- to washed red blood cells did not affect survival of Y. pestis ∆ymt in flea infections. We also investigated the hypothesis that a digestion product of serum triggers a lytic pathway in Y. pestis and that Ymt redirects or blocks this pathway. To identify Y. pestis gene products that may be altered by Ymt during infection, we subjected Y. pestis ∆ymt to random transposon mutagenesis to identify suppressor mutations. We infected fleas with pools of transposon mutants and identified one mutant that was able to survive for one week in flea guts in the absence of Ymt. The transposon insertion site was mapped to the y3762 gene, with homology to ligases (WaaL) that attach surface carbohydrate polymers to the lipid A core. Our results suggest that the WaaL modulates the outer structure of Y. pestis, enhancing susceptibility towards toxic product(s) of serum digestion. Hypothiocyanate alone is likely not the toxic agent of serum digestion. Ymt either acts on the toxic product(s) directly or inhibits the function of WaaL so that the toxic product(s) cannot enter the cell. 35. Characterization of the antimicrobial activity of the multifunctional chemokine CCL28 Bin Liu and Eric Wilson Brigham Young University The mucosally expressed chemokine CCL28 has been previously shown to mediate the migration of lymphocytes via the CCR10 chemokine receptor in vitro. Additionally, this chemokine plays an essential role in mediating the migration of IgA secreting cells in vivo. Recent research has also shown this chemokine has potent antimicrobial properties in vitro. Our research demonstrates that the highly charged C-terminal region of the chemokine is essential to this antimicrobial activity. However, the in vivo significance of this antimicrobial activity has yet to be demonstrated. We are currently generating CCL28 deficient mice to determine if the antimicrobial nature of CCL28 contributes to prevention/recovery from mastitis and other mucosal bacterial challenges. 36. A potential role for the chemokine receptor CCR9 in plasma cell homing to the mammary gland during gestation Taylor Gardner and Eric Wilson Brigham Young University The passive transfer of antibodies from mother to infant is highly dependent on the migration of Immunoglobin A (IgA) producing cells to the mammary glands. The transport and homing of immunoglobin cells is facilitated by their interactions with chemokines. The interactions of the chemokine, chemokine receptor, pair CCL28/CCR10 has been shown to play a vital role in mediating IgA ASC accumulation following parturition. However, the molecular mechanisms mediating migration of these cells before parturition have not yet been identified. In this study we investigate the role of the chemokine receptor CCR9 in mediating the migration of IgA ASC to the mammary gland during gestation. 37. A Tale of Two Salivary Glands: Differential Homing of IgA Antibody Secreting Cells Suzanne Linderman, Yuetching Law and Eric Wilson Brigham Young University While it has been proposed that the body’s mucosal surfaces have a common mucosal immune system, recent studies indicate that different subsets of IgA antibody secreting cells (ASCs) have distinct chemokine receptor repertoires which lead to specific homing to different tissues. In this study, such compartmentalization was observed in murine salivary glands where the sublingual gland contained higher mRNA and protein levels of CCL28 and its receptor CCR10, as well as 12 times more IgA ASCs per gram of tissue when compared to submandibular glands. This difference between the glands was reduced in CCR10 knockout mice and subsequently restored by adoptive transfer of wild type spleen cells to CCR10 knockout mice, indicating that

accumulation of IgA ASCs at this site is mediated by CCR10 mediated homing. Current research is aimed at determining further differences in homing to these two salivary glands, focusing particularly on the role of CCR9 which is more highly expressed in the submandibular gland, and which might therefore lead to migration of a different subset of cells. Such differences in homing are of particular interest due to the prospect of better- targeted vaccines developed with these differences in mind. 38. Buffer Requirements of CCL28 Antimicrobial Activity Kenneth Young and Eric Wilson Department of Microbiology and Molecular Biology, Brigham Young University Extensive research has been conducted in recent years concerning the role of CCL28 as a chemoattractant for cells expressing CCR10 and/or CCR3 chemokine receptors. However, little research has been done concerning the antimicrobial properties of CCL28. What research has been conducted has demonstrated antimicrobial properties of CCL28 only under certain nonphysiological buffer conditions. Previous research has not explored the chemical conditions under which this chemokine exerts its antimicrobial effect. Because CCL28 has been found in significant amounts within human saliva, milk, and mucosal surfaces of the body, a better understanding of the extent and chemical conditions of CCL28’s antimicrobial properties could lead to a greater knowledge of its role in the human immune system. Our research, testing the antimicrobial properties of CCL28 in various buffer conditions, indicates that the reduction in antimicrobial activity is based upon the osmolarity of the solution, irrespective of chemical composition. Also, it was found that an increase in CCL28 concentration was sufficient to overcome this reduction in antimicrobial activity. Therefore, it is possible that CCL28 may exhibit antimicrobial activity within discrete microenvironments of the body, where high local concentrations of CCL28 may overcome the relative inhibitory effect of physiologic salt concentrations. 39. Improved recovery of spores from coated carriers used in the AOAC Sproicidal Activity of Disinfectants (Method II), Spore Enumeration Method, by addition of a surfactant to the rinse solution. Teri M. Bills, Quin C. Shepherd, Annette J. Blam, Emily A. Moore, John Gardner, Bruce Schaalje, Kim L. O’Neill, and Richard A. Robison Brigham Young University, Utah, United States. The 2001 anthrax attacks in the United States resulted in a renewed interest in sporicidal disinfectants. Standard methods to evaluate sporicides have been in place for many years. The AOAC Sporicidal Activity of Disinfectants method uses sporecoated cylinders and suture loops as carriers. The determination of the number of spores per coated carrier was deemed important in evaluating efficacy data. Therefore, the Spore Enumeration Method was added to the AOAC procedure in 2006. This procedure involves removing the spores from carriers by sonication and vigorous mixing in water. It was theorized that the number of spores which attach to these carriers and the fraction removed may be dependent on the carrier type, the rinse solution used, and the species of spore involved. In order to evaluate these factors, removal studies were conducted using both porcelain cylinders and Dacron loops coated with one of 3 different spore species, immersed in 1 of 2 rinse solutions (water or water containing 1% Tween 80). A total of almost 300 carriers (cylinders and suture loops) coated with spores from 3 different species (Bacillus anthracis, B. subtilis, and Clostridium sporogenes) were processed in 2 different rinse fluids. Results showed significantly higher removal and recovery of Bacillus spores from both types of carriers when 1% Tween 80 was used as the rinse solution. In contrast, there was no significant effect of Tween 80 on the recovery from carriers coated with C. sporogenes spores. In addition, there was no difference in the mean number of spores recovered from rings vs. loops, coated with either Bacillus species. However, significantly higher numbers of C. sporogenes spores were recovered from rings than from loops. Exosporium differences may be responsible for the attachment and recovery disparities found between Clostridium and Bacillus spores. Because a significantly higher number of Bacillus spores were obtained when using a surfactant in the rinse solution, these data support further collaborative studies examining a revision of the Spore Enumeration Method to include 1% Tween 80 as the rinse solution. 40. Migration and Polarization of Macrophages May Be Simultaneously Affected by Macrophage Migration Inhibition Factor (MIF) B.S. Hendriksen, R.A. Enz, R. Robison, and K.L. O’Neill Department of Microbiology and Molecular Biology, Brigham Young University Current research suggests that the migration and polarization of macrophages may be necessary for the survival and proliferation of adenocarcinoma cell lines. Further studies seem to concur pointing out that macrophages make up some 70% of many types of tumors. These polarized macrophages perform different functions than the normal macrophages that perform phagocytosis and cause inflammation. The macrophages recruited by the tumors seem to promote cell healing, repair and tissue remodeling. In order to observe the effects of adenocarcinoma cell lines on macrophages we determined to use Real Time PCR to assess the protein and cytokine levels expressed. For our experiment we used U-937’s, a human mononuclear phagocyte cell line that was exposed to phorbol 12-myristate 13-acetate (PMA) to produce the macrophages. We then took these macrophages and incubated them with three different adenocarcinoma cell lines: MDA-MB-435, MDA-MB-231, and MCF7. The cell lines were separated from the macrophages by a 0.4 µm trans-permeable membrane that allowed for cell communication but not the direct contact of macrophages and adenocarcinoma cells. These macrophages and cells were then incubated for a period of 48 hours. Following this period, the expression of Macrophage Migration Inhibition Factor was measured using Real Time PCR. In MDA-MB-435 the amount of MIF increased 7.56 fold (N=9) when compared to controls, MCF7 increased 3.13 fold (N=0), and MDA- MB-231 increased 1.33 fold (N=9). Following these results we created a live cell assay to measure the migration of macrophages that correlated each cell line with the expressed MIF levels. For our migration

assay we again separated macrophages from the adenocarcinoma cell lines using a trans-permeable membrane. This time a 3.0 µm membrane was used in order to allow for active migration of the macrophages from their starting point to the adenocarcinoma cells. The macrophages and cells were then incubated for 1 hour at 37 °C. Following the incubation we compared the number of macrophages that we had started with to the number of macrophages that did not migrate during the incubation using a hemocytometer. The results were as follows: the control without cancer caused 73.86% of U937 macrophage cells to remain, MDA-MB-435 69.28% (N=24, P=0.29), MCF7 61.09% (N=24, P<0.01), and MDA-MB-231 55.83% (N=24, P<0.01). These results may suggest that MIF functions to deter the migration of macrophages that perform phagocytosis and cause inflammation while simultaneously polarizing macrophages that aide in the survival and proliferation of tumors. 41. The Potential of TK1 as a Molecular Target for Cancer Treatment. Dagoberto Estevez, Robert Whitehurst, Jaden D. Evans, Lafe T. Peavler, Melissa Tovar, Richard A. Robison, and Kim L. O’Neill Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA Thymidine kinase 1 (TK1), an enzyme responsible for recycling nucleotides in the salvage pathway, has been shown to be overexpressed in cancer cells and serum from cancer patients. To analyze the potential of TK1 as a molecular target we studied the possibility of this enzyme being expressed in plasma membranes of cancer cells. This was done with flowcytometry using a primary mouse monoclonal antibody (CB001) against TK1 and a secondary goat-anti-mouse FITC antibody. The results showed that healthy lymphocytes bound 4.03% (SD=3.75, N=3), Jurkats bound 70.17% (SD=5.40, N=9) Rajis bound 73.71% (SD=4.57, N=6.) a sample of cells from infected tonsil tissue was run and reported 8.32% binding (SD=2.01, N=3), MCF7s bound 58.63% (SD=6.00, N=9), and Hep G2s bound 77.01% (SD=10.19, N=5). Staining was then visualized using fluorescent microscopy providing evidence of TK1’s presence in plasma membranes of lymphoma cancer cells. To confirm these results we performed TK radioassays on the plasma membranes Raji cells and healthy normal lymphocytes as a negative control. Plasma membrane fractionation and separation was performed using sucrose density gradient ultracentrifugation. This assay was run with both ATP and CTP in order to calculate relative percentage values of both human isozymes: TK1 and TK2. The preliminary results showed TK1<1% and TK2>99% of enzyme activity expressed in the plasma membrane of healthy lymphocytes. In contrast, results from the plasma membrane of the Raji cells showed TK1>60% and TK2<40% of the total TK activity. Preliminary data also suggests a significant difference of total TK activity (both TK1 and TK2) in the plasma membrane of the malignant cells when compared to the healthy lymphocyte controls. These results illustrate the potential this enzyme has as a target for cancer immunotherapy. 42. Could Macrophage Phagocytosis be Regulated by the Tumor Microenvironment? D. Griffin, B. Hansen, B.R. Hendricks, R. Robison, and K.L. O’Neill. Department of Microbiology and Molecular Biology, Brigham Young University The microenvironment of most, if not all, tumors is filled with a large population of macrophages. In breast cancer, studies have found that macrophages can account for >50% of the tumor mass. However, their presence within the tumor microenvironment, in some cases, has been proven to increase metastasis, angiogenesis, and immunosuppression. For the more than 80% of cancers in which macrophages increase metastasis, angiogenesis and immunosuppression, the density of macrophages is associated with poor prognosis. Colorectal cancer is one of the exceptions that exhibits decreased tumor growth and proliferation when a larger number of macrophages are associated with the tumor. Cytokines and microbial products profoundly affect the function of macrophages. Macrophage activation in response to microbial agents and cytokines, interferon-γ (IFN-γ) in particular, has long been recognized to induce the macrophage M1 phenotype. Other studies have shown that anti-inflammatory molecules, such as glucocorticoid hormones (IL-4, IL-13, and IL-10), induce a distinct “alternative activation” or M2 activation. It has been suggested that cytokines and chemokines released by the tumor cells in the tumor microenvironment influence the polarization of the macrophages present. The M1 phenotype will often lead to aggressive destruction of the tumor, while an M2 phenotype will in fact aid tumor survival. M2 macrophages have been suggested to have a compromised phagocytic function. In this study we investigated macrophage responses to tumor products in media from various cancer cell lines. Using micosphere beads, a macrophage engulfment assay was performed using macrophages exposed to products from different types of both colon cancer and breast cancer cells. The results show that breast cancer cell products were down-regulated, or decreased, the phagocytosis by the macrophages. However, products from colon cancer up regulated the engulfment by macrophages. When macrophages are co-cultured with breast cancer cells the results strongly indicate a polarization to the M2 phenotype. This study suggests that the microenvironment significantly influences the polarization of macrophages and helps explain how tumors can evade the immune system. 43. Oxidative Stress Promotes Increased Antioxidant Uptake in a Cellular Model Jorge A. Chauca, Andrew R. Garrett, Andres D. Martinez, Evita Giraldez, Jason Loong, Jon Whitaker, José M. Peña, Richard A. Robison, and Kim L. O’Neill. Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602  Recent research has revealed a strong correlation between oxidative stress and rates of cardiovascular diseases, diabetes, and cancer. Using the Oxygen Radical Absorbance Capacity (ORAC) assay, many chemical compounds have been shown to possess high antioxidant activity. Some studies also show their effects in a cellular model. Three such antioxidants are Flavanone (140.13 TE/L), Folic Acid (676.05TE/L), and Gallic Acid (973.21TE/L). The present study examined changes in cellular antioxidant activity after exposure to each of these compounds, as well as changes after oxidative stress induced by 2,2’-Azobis (2-aminidopropane) dihydrochloride (AAPH). Raji cells (a Burkitt lymphoma line) were analyzed using the ORAC

assay to determine their baseline antioxidant activity (396.77 TE/L). Incubations with Flavanone, Folic Acid or Gallic Acid for 24 hours showed higher antioxidant activity (1229.55 TE/L, 1121.63 TE/L, and 1125.78 TE/L, respectively). Cells were also pretreated with AAPH for 15 minutes, and then exposed to Flavanone, Folic Acid or Gallic Acid. Pretreated cells with no antioxidant were used as a control. Antioxidant activity was assessed as cells recovered from stress after 10 minutes (Flavanone = 727.34 TE/L, Folic acid = 711.71 TE/L, Gallic Acid = 769.20 TE/L, and control = 822.41 TE/L), 20 minutes (Flavanone = 742.48 TE/L, Folic acid = 769.03TE/L, Gallic Acid = 717.36 TE/L, and control = 756.14 TE/L), 30 minutes (Flavanone = 881.34 TE/L, Folic acid = 1002.46 TE/L, Gallic Acid = 956.38 TE/L and control = 961.46 TE/L), and 60 minutes (Flavanone = 1532.75TE/L, Folic acid = 1508.02 TE/L, Gallic Acid = 1508.14 TE/L, and control = 1495.68 TE/L). Folic Acid was the highest antioxidant after 20 and 30 minutes of recovery. After 1 hour, Flavanone was the highest. Preliminary data indicate a steady increase in antioxidant activity as a function of time after oxidative stress. The data suggest that while cells are prone to uptake antioxidants in their environment, cell uptake increases when first exposed to oxidative stress. Further studies may also reveal optimum concentrations and times after exposure for antioxidant uptake and protection. 44. Immunolabeling Transmission Electron Microscopy: an effective method for examining location of thymidine kinase 1 in human lymphoma cells M. Hardy, M. Tovar, M. Standing, R. Robison, and K.L. O’Neill Department of Molecular and Microbiology, Brigham Young University, Provo, UT Numerous studies in recent years have demonstrated a significant up-regulation of thymidine kinase 1(TK1) in various cancer cells, giving it value as a potential therapeutic target for many types of cancer. Prior studies in our lab demonstrated the presence of TK1 on the cancer plasma membrane. Membranes from Raji cells (Burkett’s lymphoma) were isolated and assayed for TK1 activity using the TK radioassay. Results showed 60 percent more activity in the lymphoma membranes than in healthy lymphocyte membranes. The purpose of this study was to confirm these results and explore the possible pathway of TK1 to the surface of the cell through immunogold labeling transmission electron microscopy. Raji cells were fixed in formaldehyde and treated briefly with osmium tetraoxide. Sections were cut, collected on nickel grids and stained with a TK1 antibody (CB001, developed in our laboratory) and secondary antibodies conjugated to gold particles. As a negative control, grids were stained with the secondary antibody only. When observed under the transmission electron microscope, TK1 appeared within the cell membrane. TK1 appeared to follow a path from the cell nucleus to the outer membrane. This study confirms our conclusion that TK1 is present in the plasma membranes of lymphoma cells. In further studies we will examine Raji cells at various stages of the cell cycle to discover at what point TK1 is expressed in the membrane. 45. Molecular Methods for Identifying flanking sequences of Shiga Toxin genes in Escherichia coli 1 2 Zhen Li and Malcolm Shields Department of Biological Sciences, College of Arts and Sciences, Idaho State University, Pocatello, Idaho Shiga toxin-producing Escherichia coli strains (STEC) have caused increased cases of foodborne E. coli outbreaks recently. This indicates an urgent need for investigating the mechanism of STEC pathogenicity and evolution. Shiga toxins (Stx1 and 2) are the major virulence factors. The Stx genes are encoded on a lysogenic bacteriophage in the E. coli genome, which can only be expressed during the lytic cycle of the phage. Stx gene carrier phages that have been identified are lambdoid phages. However, Stx genes might also be embedded on some putative phages other than lambdoid phages. Instead of using the existing PCR protocols designed based the lambdoid phage sequences, we have developed two molecular methods to rapidly identify the flanking sequences among Stx genes: inverse PCR (iPCR) and Thermal Asymmetric Interlaced PCR (TAIL PCR). We have amplified the flanking DNA sequence of Shiga toxin genes from different Stx-producing E. coli (STEC) strains, including those isolated from castles and deer in Idaho. DNA sequencing analysis was performed to identify phage species based on amplified Stx flanking sequences. These new methods could also be used to detect Stx gene flanking sequences from environmental DNA samples, which could elucidate the possible evolutionary changes of Stx-encoding phages. 46. An Investigation of a New Predatory Bacterium (BALO) Inhabiting the Great Salt Lake, UT James W. Moyes*, Matthew J. Domek and Craig J. Oberg Weber State Univ., Ogden, UT Bdellovibrio-and-like organisms (BALOs) are small predatory bacteria that attack Gram-negative bacteria and replicate inside the host cell. There has only been one published report of BALO isolation from the Great Salt Lake (GSL) but the host organism used was a strain of marine bacteria. A soft agar overlay technique was used to screen water samples from the South Arm of the GSL against a range of halophilic bacterial hosts previously isolated from the GSL. Initial results revealed several unusual plaques characteristic of BALOs. Phase contrast microscopy of a potential GSL BALO (JM01) revealed specific BALO characteristics, such as unusual motility, very small size when compared to the host cell, and distorted spheroplasts of host bacteria that are all key indicators of a BALO. Growth and plating studies showed the formation of migrating plaques and a long lag phase for infected hosts, also indicative of a BALO infection. JM01, a potential halophilic BALO (H-BALO) showed a preference toward a single host strain, Salinivibrio S40. Inoculating halophilic broth with S40 plus JM01 resulted in a marked decrease in turbidity when compared to a control of only S40 in halophilic broth suggesting that H-BALO JM01 can inhibit Salinivibrio S40 growth in the GSL environment. 47. The Effects of Ultraviolet Radiation on Halophilic Bacteria Isolated from the Great Salt Lake Mary-Virginia Parker*, W. Dane Schofield, Matthew J. Domek and Michele Zwolinski

Weber State Univ., Ogden, UT The Great Salt Lake is a hypersaline lake in a desert environment that provides an ecological niche for many species of halophilic bacteria. Flavonoids and other pigments are considered to be protective against solar radiation due to their ability to absorb UV light. The purpose of this study was to test whether pigmented halophilic bacteria show greater survival after exposure to damaging levels of UV light compared to non-pigmented bacteria. Pigmented and non-pigmented bacteria were isolated from the Great Salt Lake and plated on standard methods agar supplemented with 8% NaCl. Halophilic bacterial isolates were identified by 16S rRNA method. Bacteria were subjected to UV-B and UV-C wavelengths for varying times and compared to bacteria not exposed to UV light. Plate counts of surviving bacteria were counted and compared to controls. Initial results suggest pigmented and non-pigmented bacteria survive moderate doses of UV light. 48. The Genome of Mycobacteriophage KLucky39 Reveals a Putative M23 Peptidase Gene. Jessica Engle, Taylor Woodward, Emily Greenhalgh, Kyler Haskell, Bryce L. Lunt, David E. Payne II Donald P. Breakwell and Sandra H. Burnett Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT. M23 peptidases are usually employed by bacteria to lyse the cell walls of other bacteria in an act of self-defense or for nutrientacquisition. A BLASTX search of a putative gene in the genome of cluster B1 mycobacteriophage KLucky39, recently isolated using the host organism Mycobacterium smegmatis, revealed a number of high-scoring matches to peptidases. Most of these were from the M23 family and were related to peptidases in genus Rhodococcus, a close relative of Mycobacterium. Exceptionally strong similarities were also found between the KLucky39 sequence and the sequences for peptidases in other B1 cluster phages PG1 and Chah. These observations provide strong evidence for identifying this open reading frame as a peptidase gene and that all similar sequences in all mycobacteriophages are also peptidases. The presence of a peptidase in mycobacteriophage sequences may indicate a potential mechanism by which this phage either enters or exits its host. 49. Addition of Novel Mycobacteriophages to Pre-existing Subclusters of the B Cluster Lissenya Argueta, Sam Petersen, Devin Sabin, Cameron Sargent, Mary Ann Taylor, Bryce L. Lunt, David E. Payne II, Donald P. Breakwell, and Sandra H. Burnett Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT. Clustering genomes based on sequence similarity provides a systematic approach for studying bacteriophage. To date, 9 clusters and 12 subclusters of mycobacteriophage have been identified using dot plot comparison and pairwise average nucleotide identity matching. Some recently discovered phages, however, have not yet been grouped or clustered. Based on genome size and gene order we hypothesized that that these phages should be grouped to into mycobacteriophage cluster B. Using Gepard, an online Java-based tool for creating dot plot comparisons of genomes, we demonstrate clustering of eight previously unclustered mycobacteriophages. KLucky39, Colbert, Fang, Puhltonio, Scoot17C, and UncleHowie clustered in the B1 subcluster; Hedgerow in the B2 subcluster; and Phlyer in the B3 subcluster. Our data suggest that phage of comparable size and gene order fit into the current system of clustering mycobacteriophage. 50. The Mycobacteriophage KLucky39 Genome Lacks Five Genes Commonly Found in Other Mycobacteriophage Subcluster B1 Genomes. Joshua Fisher, Ijesh Giri, Tambi Isaac, Bryce L. Lunt, David E. Payne II, Sandra H. Burnett, and Donald P. Breakwell Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT. Mycobacteriophage infect Mycobacterium sp. and are often found in soil. Recently, mycobacteriophage KLucky39 was isolated and its genome was sequenced. Using Gepard, an online clustering resource, KLucky39 was identified as a member of the mycobacteriophage subcluster B1 and had nearly identical gene order to other B1 subcluster phages. However, we observed that KLucky39 had five fewer genes than other B1 subcluster phages. Using BlastX and BlastN analysis of the five genes revealed that they have no obvious function in the life cycle of the phage, since they are unknown gene products. From other B1 subcluster phages we determined other potential origins of the genes: mycobacteriophages Uncle Howie and PG-1 differ from KLucky39; and in the PG-1 genome, it is evident that three different genes originated from non-B1 subclustered phage, including phage Phlyer, Phaedrus, Jasper, Gumball, Che8, Bethlehem, Ardmore, and Tweety. Finally, Uncle Howie possesses two additional genes that appear to be unique to Uncle Howie and Puhltonio. Based on our results we propose that the B1 mycobateriophages obtained these five genes from either co-infection or by mutation of preexisting genes. These observations are also further evidence of gene mosaicism in mycobacteriophage genomes. 51. Tape Measure Protein in Mycobacteriophage KLucky39 Shows Evolution of Phage Phamilies Kyle C. Smith, Michael E. Daetwyler, Zach S. Liechty, Michael C. Severson, Brigham Wright, Bryce L. Lunt, David E. Payne II, Donald P. Breakwell, and Sandra H. Burnett Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT. Most mycobacteriophages are classified in Siphoviridae, a bacteriophage family characterized by their long tails. In all mycobacteriophages, tape measure proteins, associated with the synthesis of tail proteins, are present. The length of this tape measure protein is correlated with the length of the phage tail and allows for a comparative analysis of the relationships and divergence of this phage phamily. Comparing the tape measure proteins in KLucky 39, a mycobacteriophage recently isolated in Utah, to other mycobacteriophages identified possible genetic variations that have lead to the evolution of differences in this

phage phamily. Using BLASTx sequence analysis of the KLucky39 gene sequence, we found relationships with B1 phage subclusters with 98% covered identities with no gaps. In mycobacterium phages Cooper, Nigel, Phlyer, Phaedrus, and Piperfish, from subclusters B2, B3, and B4 we found similarities ranging from 41-71% identity with two interesting nonhomologous segments. Variation suggests that between the tape measure proteins of different phages, phylogeny and evolutionary relations are illustrated. 52. Mycobacteriophage Clusters Exhibit Discrepancies in the Distance of Shine-Dalgarno Sequences from the Start Codon Adam Hansen, Kara Ladle, Alex Benedict, Bryce L. Lunt, David E. Payne II, Sandra H. Burnett, and Donald P. Breakwell Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT. Mycobacteriophage infect bacterial hosts within the genus Mycobacterium. A number of computer programs exist to aid in genetic annotation. One of these programs— “Shine-Dalgarno Finder”— identifies putative Shine-Dalgarno (SD) sequences by assigning numerical scores to each nucleotide sequence, and by comparing them to a consensus SD sequence. As identified by SD Finder, significant discrepancies exist in the distances between the location of putative SD sequences and start codons of putative genes from one phage cluster (B1 – KLucky39) to the next (B2 – Rosebush, Hedgerow). Identification of these potential patterns and discrepancies reveal differences between phage clusters. Recommended changes may further improve upon the program and more accurately predict SD sequences. 53. Comparison of KLucky39 Gene 41939 with Known E. coli Genes Kyler Haskell, Pradip Bajgain, Brian Earley, Bryce L. Lunt, David E. Payne II Donald P. Breakwell, and Sandra H. Burnett Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT. KLucky39 is a subcluster B1 mycobacteriophage, a group of phage infecting Mycobacterium sp. During annotation of this genome, using BLASTX, we showed that a KLucky39 gene was related to several bacterial genes, including those found in Escherichia coli. Specifically we observed homology to proteins in different strains of E. coli including a putative prophage protein (E. coli O103:H2) E = 2e-25; a short tandem repeat (two genes total) of bacteriophage P4 DNA primease (E. coli O157:H7) E = 2e-25; a putative DNA primase (E. coli O26:H11) E = 2e-24; and bacteriophage P4 DNA primease (E. coli E110019) E = 6e-23. Also associated with this putative gene product was similarity to bacteriophage P4 DNA primease and also a putative primase/primease. Since prophages are lysogenic, this gene may regulate KLucky39 replication and lysis of host bacteria. These processes can be interrelated and thus may encode for both lysogenic and replication/transcription. This observation increases our understanding of these genes in the life cycle of mycobacteriophages. 54. Characterization of an Arabidopsis nuclear-encoded gene (TWINKLE) for a homologue of the bacteriophage T7gp4 protein John Cupp and Brent Nielsen, Brigham Young University Plant mitochondrial genomes are large, complex, and may replicate by a recombination-dependent mechanism or by more than one mechanism. We have identified an Arabidopsis nuclear-encoded gene for a homologue of the bacteriophage T7gp4 protein that has both DNA primase and DNA helicase activities. The plant homologue has high homology with the T7 protein, while the animal homologue (Twinkle) lacks homology over the DNA primase domain and has been shown to have DNA helicase but not primase activity. In animals this protein controls mtDNA replication and genome copy number. We have shown that this protein in Arabidopsis is localized specifically to mitochondria and that it has both DNA primase and DNA helicase activities. Homozygous T-DNA insertion lines show severely reduced growth rates and premature flowering with significantly lower seed production. Confocal microscopy analysis indicates significant alterations in mitochondrial/nuclear organization in root tips such as a reduction in mitochondrial density. Quantitative real-time PCR analysis shows a reduction in mtDNA levels in the mutant lines. These results suggest that this protein is necessary for mtDNA replication and normal plant development. 55. Isolation of Novel Phage from the Great Salt Lake for Idiomarina-like Bacteria C. M. Benson, M. J. Domek, M. D. Zwolinski, and C. J. Oberg Microbiology Department, Weber State University, 2506 University Circle, Ogden, UT, 84408 Characterization of halophilic bacteria from the South Arm of the Great Salt Lake (GSL) revealed a number of Idiomarina-like organisms. Since phage can play a role in controlling bacterial populations in the environment and facilitate lateral gene transfer, water samples from the South Arm were collected and filtered to remove bacteria. The filtrate was tested against four Idiomarina strains from the GSL to look for phage specific to this genus. Initial screening showed that a large number of plaques developed with a wide range of sizes. One group of plaques formed a 10 mm clear zone with a much larger opaque halo extending around each plaque. A second group of plaques were approximately 2 mm in diameter. A third plaque morphology was the smallest, only a one mm diameter. The fourth group of plaques had variable shapes. The plaque morphologies appeared consistently on host strains, as individual phage were isolated, purified, and propagated. A host comparison was performed with purified phage isolates against the four strains of the Idiomarina to further characterize the phage isolates. Based on host range results, five phage types were identified based on their host specificity. The first type of phage (phage 11, 12, 15, and 16) only infected Idiomarina isolate S3. The second type (phage 2, 3, 6, 10, 13, and 14) could infect two different hosts, Idiomarina S3 and S11. The third type (phage 9) infected Idiomarina S3 and S21 while a fourth phage type (phage 8) only infected Idiomarina S21. The fifth type of phage (phage 1, 4, 5, and 7) was found to infect only Idiomarina S11. Currently, no phage have been found that infect Idiomarina S6. The 16S rRNA gene sequences of the Idiomarina-like isolates had less than 0.5% difference over 1,300 bp

indicating that phage typing may provide greater strain resolution than 16S rRNA gene sequencing. Further, there appears to be some correlation between plaque morphology and host range. 56. Prophage Induction in Halophilic Bacteria Isolated from the Great Salt Lake Karli Oberg, Karen Nelson, Christie Jensen, Trever Gray, Adam Hutchinson, Matt Domek, and Craig Oberg Weber State University, Ogden, UT Limited research has been performed on lysogenization in halophilic bacteria found in moderately halophilic environments. Thirteen halophile bacteria cultures were isolated from the South Arm of the Great Salt Lake (GSL). Broth cultures of these isolates were exposed to either ultraviolet (UV) radiation at various time intervals or to mitomycin C at various concentrations to initiate prophage induction. A comparison between untreated control cultures and UV-treated cultures showed much slower growth in treated tubes over time, denoting prophage induction. Some of the UV-treated culture tubes showed the characteristic drop in OD600 after several hours of incubation, also indicative of prophage induction. Of note, many of the halophilic strains were very sensitive to the UV treatments, surprising since these strains are exposed to UV light in their environment. The standard mitomycin C concentration utilized for prophage induction of 0.5 µg/ml had no effect on six GSL cultures used for the mitomycin C induction study. Three strains of Salinivibrio (S9, S38, and S50) showed characteristic induction growth curves at a mitomycin C concentration of 1.5 µg/ml while on Idiomarina-like strain (S3) exhibited prophage induction at a concentration of 1.0 µg/ml. Several of the GSL cultures, including a Salinivibrio (S40) and an Idiomarina-like strain (S21) exhibited no response to any of the mitomycin C concentrations used, except an increased lag phase at 1.5 µg/ml. Spot tests with a soft agar overlay using filtrates from mitomycin C treated cultures also suggested that prophage have been induced from several strains These results suggest the existence of prophage in some halophile isolates and indicate higher concentrations of mitomycin C than normally utilized can cause prophage induction. 57. Survey of Chitinase Activity in Halophilic Bacteria Isolated from the Great Salt Lake Jared T. Phelps, Craig Oberg, Michele Zwolinski Microbiology Department, Weber State University Chitin from brine shrimp and brine fly exoskeletons is an important source of nitrogen and carbon in the Great Salt Lake (GSL). Microorganisms depolymerize chitin using a combination of chitinase enzymes that cleave the bonds between N-acetyl-Dglucosamine (GlcNAc) residues, resulting in GlcNAc monomers or short oligomers that can be transported into the cell. Although chitinase activity is widespread among microorganisms, little is known about the distribution or diversity of chitindegrading halophiles. A collection of bacterial GSL halophilic isolates, representing five genera, were assayed for exo- and endochitinase activity. Isolates were incubated with fluorescently labeled chitin analogues with one (monomer), two (dimer), or three (trimer) GlcNAc residues. Cleavage of the label from the chitin analogues resulted in fluorescence that was measured with a spectrophotometer. Among the isolates tested, the chitinase patterns were genus specific. The Salinivibrio-like organisms were able to cleave all three GlcNAc substrates, indicating exo- and endochitinase activity. These organisms cleaved the monomer after a short incubation, indicating the presence of the exochitinase in the culture medium. The dimer and trimer were depolymerized more slowly which could indicate these enzymes are induced by the two substrates. The Bacillus and Marinicoccus-like isolates only cleaved the monomer and may depend on organisms such as the Salinivibrio for access to the GlcNAc monomer. Although chitin is a large source of carbon and nitrogen in the GSL, Idiomarina-like organisms isolated from the lake did not show significant use of any of the substrates. This finding is supported by genomic analysis of other Idiomarina organisms. Using this assay, several organisms capable of producing salt tolerant chitinases have been identified. Further, characterizing the chitinase profiles of GSL organisms helps clarify the ecology of the major nutrient cycles in the lake.